The purpose of the proposed research is to utilize the duckling salt gland as a model system for the study of the mechanism of plasma membrane biogenesis and removal. The duckling salt gland is advantageous for these studies because plasma membrane amplification and then removal can be induced at will by first osmotically stressing the ducklings by feeding salt water and subsequently reversing the stress by feeding fresh water. This is observed by electron microscopy. A time course for the initiation of both synthesis and removal of plasma memrane components is being established by measuring changes in the rates of incorporation of protein, glycoprotein, and lipid pecursors during both processes. The major specific plasma membrane protein marker used is the Na,K-ATPase which is measured by ATP hydrolysis, quantitated by 3H-ouabain binding and localized by cytochemistry or 3H-ouabain autoradiography. Pulse chase autoradiography experiments, using 3H-precursors of membrane glycoproteins (fucose) and lipids (glycerol) to trace the route of plasma membrane assembly are employed. Electron microscopy reveals a prominent golgi, transfer vesicles and numerous free polysomes that may be involved in biogenesis, and numerous acid phosphatase-rich, cytoplasmic vesicles that may be involved in removal of plasma membrane. A plasma membrane fraction from salt gland will be prepared and examined by disc gel electrophoresis and double labelling techniques to determine the half lives and changes in membrane protein profiles during biogenesis and removal of plasma membrane components. Advantage is also being taken of the affinity of ouabain for the Na,K-ATPase, to develop cytochemically useful derivatives of ouabain for localization of the Na,K-ATPase. A conjugate of ouabain-microperoxidase has been developed and utilized and ouabain-diamohexane is being synthesized. The latter can be covalently linked to the Na,K-ATPase for E.M. autoradiography applications, or conjugated to ferritin or cytochemically useful reagents.